KR101456205B1 - Immunomodulatory and anti-tumour peptides - Google Patents

Abstract

The present invention relates to the improvement of peptides derived from the sequence HYRIKPTFRRLKWKKYKGKFW in which amino acid substitutions are introduced to insure separation of lipid polysaccharide-binding capacity and increase anti-tumor and immunomodulatory effects. The peptides and combinations thereof can be used for synergistic effects with treatment of cancer as well as with other standard treatments.

Anti-tumor peptide, LPS, heparin

Description

Immunomodulatory and anti-tumor peptides {IMMUNOMODULATORY AND ANTI-TUMOR PEPTIDES}

The present invention is included in the field of cancer immunotherapy. More precisely, a peptide derived from SEQ ID NOS: 32-51 of the Limulus anti-LPS factor protein, or a combination thereof, is capable of binding to lipid polysaccharides and is useful for the treatment of cancer and metastasis.

Recent therapeutic therapies for enhancing the benefits of therapy and the use of modulators of biological response to treat cancer in combination, have been recently reported (US 2004/0101511). On the other hand, the use of the CpG sequence, an action of the Toll-like receptor 9 (TLR9), has been implicated in the treatment of cancer, non-small cell lung cancer, (Klinman DM, et al, (2004) Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol . 4: 249-258). Toll-like receptor 7 (TLR7) agonists have now been tested in Phase I clinical trials to activate the immune system, expecting results as novel drugs for treating melanoma and other tumors (Dudek AZ, et al ASCO Annual Meeting). The above-mentioned agonists of TLRs 7 and 9 have also been evaluated in viral infections based on their ability to stimulate an effective immune response in the host. In addition, named heat shock proteins (Hsp) that bind to TLR4 have been improved and prepared as fusion proteins to human papillomavirus (HPV) E7 oncoprotein. This novel immunotherapeutic approach is also known as a therapeutic vaccine having a broad perspective for treating human papillomavirus-associated diseases (Chu NR et al., (2000) Immunotherapy of a human papillomavirus (HPV) type 16 E7 -expressing tumor by administration of fusion protein comprising Mycobacterium bovis bacille Calmette-Guerin (BCG) hsp65 and HPV16 E7. Clin Exp Immunol 121: 216-225). Toll-like receptors are receptor molecules present in the cells of the immune system that recognize pathogen-associated molecular patterns, such as LPS, lipoteichoic acid, unmethylated CpG sequences and viral double- and single-stranded RNA. Recognition of pathogen invasion by TLRs aids the immune system causing a balanced Th1 / Th2 immune response to effectively exterminate external infections of the organism. The use of TLRs actives as drugs for the treatment of cancer is driven by the activation of the Th1 immune response mediated by interleukin 12 (IL-12) and interleukin 12 (I < RTI ID = 0.0 > immune < / RTI > and adaptive immune system. Thus, the immune response is made as to be kept highly specific and (Switaj T., Jalili A., et al., (2004) CpG Immunostimulatory oligodeoxynucleotide 1826 enhances antitumor effect of interleukin 12 gene-modified tumor vaccine in a melanoma model in mice. Clinical Cancer Research , Vol. 10: 4165-4175). This dual activation of the immune system contrasts with several other immunotherapeutic approaches that do not produce sustained effects in adaptive immune responses with sequential undesirable effects (Speiser D. E, et al. strong human CD8 + T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909. The Journal of Clinical Invest . Vol. 115 (3)).

Dendritic cells (DCs) are specialized antigen presenting cells involved in innate and adaptive immune responses through cell-to-cell interactions and cytokine production. DCs are divided into a series of surface molecule markers and also lymphoid cells which are based on differentiated expression of TLRs, myeloids or lymphocytes originating from them. Lymphocyte DCs, also known as plasmacytoid DCs, are a major source of type I interferons. Considering these characteristics, DCs have been engineered to be expected as cellular agonists to improve the therapeutic vaccine against cancer and chronic viral infections (Santini SM, et al (2003) A new type I IFN-mediated pathway for rapid differentiation of monocytes into highly active dendritic cells . Stem Cells , 21: 357-362). However, this is a very expensive and difficult technique that involves other more practical and less costly therapeutic strategies under improvement (Van Epps HL (2005) New hope for tumor vaccines. The Journal of Experimental Medicine , Vol. 202: 1615).

Type I interferons (a, b IFNs), originally described by their antiviral activity, exhibit an important effect across the immune system, promoting cellular and humoral immune responses through their adjuvant systems in DCs (Bogdan, C. (2000) The function of type I interferons in antimicrobial immunity. Curr , Opin Immunol . 12: 419-424). Recent work has clarified the clinical role of endogenous type I interferon in the pathway of degeneration of highly immunogenic synergistic mouse sarcoma and in the host's protection against the development of primary carcinogenic tumors (Gavin P. Dunn, et al. ) A critical function for type I interferons in cancer immunoediting. Nature Immunology , June 12). In addition, IFN-a plays an important role in initiating antiviral T-lymphocyte responses through direct activation of CD4 + or CD8 + T lymphocytes in viral infections such as influenza (Fonteneau JF, et al. (2003) Activation of influenza virus -specific CD ⁺ 4 and CD ⁺ 8 T cells: a novel role for plasmacytoid dendritic cells in adaptive immunity. Immunobiology , 101: 3520-3526).

Hoess et al. (WO 95/05393) discloses a method of treating a subject afflicted with LPS that has high affinity, useful for treating or preventing an infection such as gram-positive or gram-negative bacterial-mediated sepsis, general bacterial infections and fungal infections. (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A ⁰ resolution. The EMBO J. 12: 3351-3356). The crystal structure of the original Limulus anti-LPS factor (LALF) protein represents a loom similar to polymyxin B, including positively altered amphiphilic and hydrophobic and aromatic residues. Based on this principle, the ability of the sequence corresponding to amino acids 31-52 in the LALF protein to neutralize and bind heparin binding effects such as anti-coagulation, angiogenesis and inhibition of endothelial and tumor cell proliferation in this document . However, there is no experimental data to support such a reference in the above mentioned patents. In fact, the approved claims refer to a device for removing LPS from solution, which comprises immobilized peptides in a solid support (US 6,384,188).

On the other hand, the literature Vallespi (US 6,191,114) relates to the antiviral effect of the LALF _{31 -52} peptide on Hep-2 and MDBK mediated by the production of α and γ interferons, and viral infections and immunosuppression-related disorders And to the use of this peptide for treatment. In addition, the same authors have demonstrated the anti-infective effect of this protein in animal models of sepsis (Vallespi MG et al (2003) A Limulus anti-LPS factor-derived peptide modulating cytokine gene expression and promoter resolution of bacterial acute infection in mice. International Immunopharmacology , 3: 247-256).

There are many treatments directly to cancer, including chemotherapy, radiation and gene therapy. Toxicity is a major drawback of all these treatments and has been administered for extended periods of time, which ultimately results in some therapeutic effect at high doses. Therefore, the development of new drugs for obtaining more effective treatments is still required.

Based on the basic role of the immune system to detect and direct an effective response to a tumor, drugs designed to activate the host native and adaptive defense mechanisms become a powerful tool for cancer therapy.

There was no previous amino acid substitution described for the sequence HYRIKPTFRRLKWKKYKGKFW of the LAF protein, which removes the LPS-binding ability and enhances the immunomodulatory effect and also confer in vivo anti-tumor effects on multiple tumors.

The present invention provides peptides derived from the 32-51 region of the LALF protein sequence HYRIKPTFRRLKWKKYKGKFW (SEQ. ID. NO: 13) to solve the above-mentioned problems, wherein amino acids eradicate LPS- And to enhance immunomodulatory effects.

Analogous peptides derived from such sequences by substitution that interfere with LPS- or heparin-binding and also provide increased anti-tumor and immunomodulatory effects relative to the original peptide consist of the following sequence:

H A RIKPTFRRLKWKYKGKFW (SEQ ID NO: .. 1)

HYRIKPT A RRLKWKYKGKFW (SEQ ID NO: .. 2)

HYRIKPTFRRL A WKYKGKFW (SEQ ID NO: 3)

HYRIKPTFRRLKWKYKGKF A (SEQ ID NO:. . 4)

The LPS-binding peptides described by Hoess and Vallespi are disadvantageous, deviating from the mixed Th1 / Th2 profile directed against the predominant, predominant Th2 profile, which is disadvantageous to cancer patients. These patients have an accompanying infection due to immunosuppression, not excluding the presence of LPS particles in the blood. Administration of a peptide comprising a predominant Th2 profile in the presence of LPS will cause side effects that lower the patient ' s immunological status and exacerbate the host response to the tumor. In addition, binding of the peptides described by Vallespi to LPS will minimize the immunomodulatory effect of these peptides.

On the other hand, the lack of binding of the peptides described in the present invention to heparin makes it better than previously described by Hoess and Vallespi. In actual patients, such as patients with ischemic heart disease, cerebral vascular disease, venous thromboembolic disease (deep vein thrombosis and pulmonary embolism), and patients with hindlimb ischemia, heparin appears as a therapeutic agent (D . Cabestrero Alonso , et al (2001) Heparinas de bajo peso molecular en pacientes criticos: usos, indicaciones y tipos. Medicina Intensiva , Vol. 95: 18-26). Thereafter, the administration of the heparin-binding peptide will exhibit taboos in this context, because it interferes with the effect of this drug. The association of cancer and thromboembolism is a well-known symptom that can significantly contribute to morbidity and mortality in cancer patients, such as deep vein thrombosis and pulmonary embolism. Based on the above-mentioned reasons, the efficiency of the use of peptides, which inhibit binding to heparin and exhibit anti-tumor and immunomodulatory effects in the treatment of cancer patients, (Castelli R., et al. (2004) The heparins and cancers: Review of clinical trials and biological properties. Vascular Medicine , Vol. 9: 1-9).

The present invention also includes peptides having two or more amino acids substituted by alanine, including the following amino acid sequences:

HYRIKPT A RRL A WKYKGKFW (SEQ ID NO:.. 8)

H A RIKPT A RRLKWKYKGKFW (SEQ ID NO:.. 9)

H A RIKPTFRRL A WKYKGKFW (SEQ ID NO:.. 10)

H A RIKPT A RRL A WKYKGKFW ( SEQ ID NO:.. 11)

H A RIKPT A RRL A WKYKGKF A (SEQ ID NO:.. 12)

As part of a fusion peptide and other homologous or analogous variants of such previous peptides obtained by synthetic or recombinant processes. Similar variants represent all peptides that lack LPS- or heparin-binding ability and exhibit anti-tumor and immunomodulatory effects. Similarly, the analog variants are represented by all molecules of a chemical origin (non-protein) whose structure lacks LPS- or heparin-binding ability and maintains anti-tumor and immunomodulatory effects.

In a preferred embodiment of the invention, the pharmaceutical compositions comprise one or more peptides and chemical compounds or pharmaceutically acceptable salts thereof, as well as pharmaceutically acceptable excipients or excipients.

In another preferred embodiment of the invention, the pharmaceutical composition further comprises an antigen selected from the group comprising bacterial, viral or cancer antigens.

Similarly, the peptides of the invention may be used in combination with conventional therapies for cancer, such as chemotherapy, surgery, radiation, and the like.

The invention also relates to the use of such peptides and chemical compounds for the manufacture of a pharmaceutical composition for the treatment and / or prevention of an immunological disorder in which an effective Th1 immune response is required; Lt; RTI ID = 0.0 > cancer, < / RTI > which improves an effective immune response to an infection of bacterial or viral origin.

The described peptides are defined as lacking the LPS-binding ability, instead of the original sequence, which has been described previously as the common optimal domain for LPS binding (Hoess et al., (1993) Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5A ⁰ resolution. The EMBO J. 12: 3351-3356). In the same way, the peptides described in the present invention can be used as peptides, as compared to peptides derived from LALF proteins comprising amino acids 31-51, which Vallespi refers to as an antiviral and immunomodulatory peptide in his patent (US 6,191,114) Enhances the immunoregulatory effect mediated by the secretion of IFN- [alpha].

Similarly, the peptides described in the present invention can be administered to immunosuppressed patients in need of activation of their immune status, such as patients suffering from acquired immunodeficiency syndrome (AIDS) and patients undergoing combined surgery .

Experimental in vivo data demonstrate the efficacy of the peptides in tumors implanted in mice, which are murine melanoma B16 cells; Derived from 3LL-D122 cells obtained from malignant lung epithelial cells and mouse lung cancer from C57Bl / 6 mice.

In other embodiments, the peptides can be administered to minimize metastatic events.

Other results of the present invention indicate that the peptides described exhibit anti-proliferative effects in tumor cell lines of various histological origins, demonstrating a direct cytotoxic effect across cancer cells.

In principle, the peptides described can be used alone or in combination with therapies for treating cancer, such as surgery, radiation or chemotherapy.

Similarly, the peptides described in the present invention, when prophylactically administered, produce a rapid native immune response against the tumor, resulting in an adaptive antigen-specific immune response and emphasize their use in prophylactic or therapeutic vaccines against cancer did.

Figure 1: Effect of peptides on bacterial lipopolysaccharide (LPS) -binding capacity. 1a, these experiments are in triplicate; The inhibition curve of one experiment is shown. The percent inhibition shown in Figure 1B represents the average of three independent experiments.

Figure 2: Peptide effect in the ability to bind an anionic compound heparin. It represents the average of three independent experiments.

Figure 3: Effect of peptides on the production of alpha and gamma interferon and IL-12 in human mononuclear cells. The three experiments were composed of other donors that appeared to cause one of the above.

Figure 4: Anti-tumor effect of peptides in TC-1 tumor model.

Figure 5: Anti-tumor effect of L-2 peptide in melanoma model.

Figure 6: Anti-tumor effect of similar L-2 peptides in prophylactic dosing schedules of TC-1 tumor models.

Figure 7: Anti-tumor effect of a similar L-2 peptide on a dual dosing schedule of the TC-1 tumor model.

Figure 8: Anti-melanoma potential of similar peptide L-2.

Figure 9: Effect of the similar peptide L-2 on proliferation of TC-1, H125 and L929 cells.

Example 1. Peptide synthesis

The peptide of the present invention was synthesized according to a solid-phase process. The unpurified peptide was extracted with 30% acetic acid solution, lyophilized, and further purified by RP-HPLC. The molecular weight of the purified peptide was confirmed using a mass spectrometer JEOL JMS-HX110HF with FAB gun. The resulting formulation is non-pyrogenic and is pharmaceutically acceptable for administration to animals and humans. Substitution was performed by inserting an alanine amino acid at each position of the original sequence of the peptide

Example 2. Selection of Lipopolysaccharide (LPS) -Substituted Peptides Without Binding Ability.

This test is constructed in a competitive system (Hardy E., et al. (1994) Enhanced ELISA sensitivity using TCA for efficient coating of biologically active LPS or Lipid A to the solid phase. J. Immunol . Meth . Vol.176: 116). Polystyrene plates (Costar, USA) were inoculated into E. coli with 0.2% trichloroacetic acid (TCA) 0111: LPS from B4 (1 [mu] g / ml). Plates were incubated overnight at 37 ° C and washed 10 times with 1X phosphate-buffered saline (1X PBS) (wash solution) supplemented with 0.1% Tween-20. Binding of the similar peptide to the LPS immobilized on a solid surface was evaluated by competitive ELISA with biotinylated LALF _32-51 at 0.2 [mu] M and exhibited a maximum LPS binding of 90%. To measure the inhibition curve, we used the peptide of the other similarities concentration of 10 μM to 0.01 μM were used as an experimental curve of the LALF _{32 -51} contrast. Was biotinylated LALF _32-51 during the two hours at constant temperature and in the presence of 37 ℃ similarities peptide and the plates washed five times with wash solution. The biotinylated binding to LPS LALF _{32 - 51} 1 - was at 37 ℃ with the peroxidase conjugates with a constant temperature is detected for 45 minutes at 2,000 dilution of streptavidin. The plates were washed five times with washing solution and substrate solution was added (0.05 M Citrate-Phosphate Buffer, pH 5.5, 1, 3, 5, 5 'tetramethylbenzidine and one 025% hydrogen peroxide purification). After 15 minutes of incubation, the reaction was stopped by the addition of 2 M sulfuric acid. Absorbance was quantified at 45 nm in a plate reader (Sensident Scan). There is a correlation between LPS-binding ability and optical density values of similar peptides. No similarities peptide having a high LPS- binding capacity show inhibition curves of lower OD more than the inhibition curve of the LALF _{32 -51} peptide.

Similarity of a peptide having a low LPS- binding capacity represents the inhibition curve in the OD value than the inhibition curve of the LALF _{32 -51} peptide. The results showed in Fig. 1a, which L-2, L-8, the peptide named as L-12 and L-20 is similar to the LALF _{32 -51} peptide, peptides having L-9 and L-19, which retains the ability , Demonstrating the loss of ability to bind LPS. On the other hand, L-3 peptides exhibit higher LPS binding capacity.

Figure 1b is in accordance with their ability to displace the binding of the biotinylated LALF _{32 -51} (0.2 μM) to LPS adsorbed to the solid surface, it shows the inhibition percentage of similarity of peptide at a fixed 0.5 M concentration.

Inhibition% = {1 - ((OD) _sample - [OD] _min ) / ([OD] _max - [OD] _min ))} x 100

[OD] _Samples : optical density values in the presence of fixed concentrations of similar peptides;

[OD] _Minimum : ELISA background;

[OD] _max : Optical density figure without peptides.

As a result of this assay, double, triple, and quadruple mutations have occurred starting from peptides lacking LPS-binding ability:

HYRIKPT A RRL A WKYKGKFW (SEQ ID NO:.. 8)

H A RIKPT A RRLKWKYKGKFW (SEQ ID NO:.. 9)

H A RIKPTFRRL A WKYKGKFW (SEQ ID NO:.. 10)

H A RIKPT A RRL A WKYKGKFW ( SEQ ID NO:.. 11)

H A RIKPT A RRL A WKYKGKF A (SEQ ID NO:.. 12)

Example 3: Evaluation of heparin-binding ability of similar peptides L-2, L-8, L-12 and L-20.

This assay was constructed on a competitive ELISA system similar to that described above. Biotinylated LALF _32-51 peptide was adsorbed onto polystyrene plates (Costar, USA) in 1X PBS and incubated overnight at 4 ° C. (Sodic Heparin, 5,000 U / ml, Liorad) in 1X PBS supplemented with 0.1% bovine serum albumin (BSA) At 2 [mu] M. Thereafter, the mixture was added to the ELISA plate containing the biotinylated LALF _{32 -51} peptide adsorbed to a solid surface of 0.02 μM. After a constant temperature for 1 hour at room temperature, the plate washed 5 times with the washing solution, and fixed to a solid surface biotinylated LALF _{32 -51} peptide 1: 2000 in Streptavidin-peroxidase at 37 ℃ with a constant temperature 45 minutes . The plate was then washed five times with wash solution and substrate solution added. After an additional 15 minutes of incubation, the reaction was quenched with 2 M sulfuric acid solution. The lack of heparin binding to the similarities peptides because they can not be substituted for the heparin binding to the biotinylated LALF _{32 -51} peptide adsorbed to a solid surface, are associated with one another with a reduced optical density. An unlabeled LALF _32-51 peptide at a 100 X molar excess was used as the control for the assay. Heparin Binding of unlabeled peptide LALF _{32 -51,} so the amount of excess of cold peptide competition due to the binding of heparin, was demonstrated by the increased optical density value. As shown in FIG. 2, the results indicate that the peptides L-2, L-8, L-12 and L-20 of the present invention can not bind to heparin.

Example 4. Effect of peptides on expression of IFN-a, IFN-y and IL-12 in human mononuclear cells.

In this assay, human mononuclear cells were isolated by leukocyte concentrate or "Buffy Coat" of the donor by Ficoll-Hypaque gradient. Were inoculated into 24-well plates in RPMI 1640 medium supplemented with 10% calf serum to 5x10 < ^{6 &} gt ;. Each peptide was added at 40 [mu] g / ml in 0.1 ml volume of RPMI medium and the cells were incubated at 37 [deg.] C and 5% of CO ₂ for 18 hours. Total RNA was extracted using the TriReagent method. The expression of the expressions of IFN-α, IFN-γ and IL-12 was then determined by reverse transcription and by PCR amplification (RT-PCR kit, Perkin Elmer). The results are expressed as the relative amounts of the generalized messenger RNA for expression levels of the b-actin housekeeping gene. The results obtained in this assay show that the peptides L-2, L-8, L-12 and L-20 described in the present invention exhibit the expression of IFN- ?, IFN-? And IL- Proving that it can be induced. Similarity of peptide L-2, L8 and L-12 are particularly more effective in the induction of IFN-α expression of the gene than the LALF peptide _{32 -51} as shown in Figure 3b in particular. This embodiment eliminates the ability of the amino acid substitution in the binding to LPS aid LALF _{32 -51} sequence, enhance the immunomodulatory effect of the resulting peptide as such.

Example 5. Anti-tumor effects of the similar peptides L-2, L-8, L-12 and L-20 in the TC-1 tumor model.

8-10 week old C57Bl / 6 female mice were used in this assay (n = 10 animals per experimental group). In tumor grafts, TC-1 cells derived from C57B1 / 6 malignant epithelial lung cells were suspended in phosphate-buffered saline (PBS). The amount of 50,000 cells in a volume of 200 [mu] l was subcutaneously inoculated into the right hind leg of the mouse. The first peptide was administered via the subcutaneous route in the right flank when the tumor reached 100 mm ³ volume, and the second peptide was administered 10 days later. In this assay, a dose of 4 mg per kg of weight was evaluated (80 ug / mouse). The animal survival rate and tumor size were evaluated to determine the anti-tumor effect of the peptides of interest as shown in Figures 4a and 4b. Similar peptides L-2, L-8, L-12 and L-20 are effective in inhibiting tumor progression and prolonging survival in mice, respectively. These results demonstrate the anti-tumor efficacy of the peptides in a fidelity tumor model of mice. The Log Rank method was used as a statistical analysis for the significant difference between groups. The results demonstrate that the similar peptides L-2, L-8, L-12 and L-20 significantly increase animal survival compared to LALF _32-51 peptide ( ^* p <0.05). These results demonstrate that substitution of the amino acid in the original 32-51 sequence of the LALF protein can significantly increase the anti-tumor ability of the peptide.

Example 6. Anti-tumor effect of comparative peptide L-2 in melanoma model.

C57Bl / 6 female mice between 8 and 10 weeks of age were used in this assay (n = 10 animals per experimental group). With tumor graft, MB16-F10 tumor cells were resuspended in phosphate-buffered saline (PBS). The amount of 15,000 cells was inoculated into the right hindpaw via a subcutaneous route with a volume of 200 μl. After 4 days, the L-2 peptide was administered via secondary infusion administered 7 days after primary administration and tertiary immunization administered 14 days thereafter. In this assay a dose of 4 mg / kg animal weight was evaluated. The parameters evaluated to determine the anti-tumor effect of a given peptide during this assay are the time of tumor transplantation and the survival rate of the animal. As shown in Figure 5a, L-2-like peptides significantly delayed tumor transplantation ( ^* p < 0.05, log-rank method). Survival analysis by the log-rank method was demonstrated ^(* p <0.05), compared to the LALF _{32 -51} peptide more efficiently-rescue a significant increase in the survival of animals, as shown in the L-2 peptide Figure 5b. These results demonstrate anti-tumor efficacy against cancer cells from other histological and anatomical parts of the body, such as lung epithelial cancer cells as well as melanoma.

Example 7. Anti-tumor effect of L-2-like peptide in prophylactic schedule of treatment in TC-1 tumor model.

C57Bl / 6 female mice between 8 and 10 weeks of age were used in this assay (n = 10 mice per experimental group). With tumor grafts, TC-1 tumor cells were resuspended in phosphate buffered saline (PBS). Mice were firstly injected with L-2 peptide (4 mg peptide per kg of body weight). Seven days later, mice were injected with the same dose twice; Fourteen days after the primary injection of the peptide, the animals were inoculated with 50,000 cells in a volume of 200 [mu] l by the subcutaneous route in the right hind limb. The parameters were evaluated to determine the anti-tumor effect of the peptide including tumor transplantation time (Figure 6a) and animal survival (Figure 6b). The results obtained in this assay demonstrated that the L-2 peptide of the present invention is effective in preventing the development of the tumor and also increasing the survival rate of the animal. These results demonstrate that the peptides of the present invention exhibit prophylactic effects of preventing the establishment of tumors.

Example 8. Anti-tumor effect of L-2-like peptide in dual antigen dosing schedules.

To the tumor-free animals (n = 6) established in the schedule of the above example, 50,000 TC-1 cells in a volume of 200 [mu] l in PBS were subcutaneously administered subcutaneously in the left hind paw (49 days after primary antigen challenge ). To ensure homogeneity at age, equal volume of tumor cells were inoculated into the same "untreated" mice as a control group of experiments and maintained under the same conditions (n = 10 animals) without administration of the peptides. Parameters were evaluated to determine the anti-tumor effect of L-2 peptide, including tumor transplantation time and animal survival. The results obtained in this assay demonstrate that the L-2 peptide can block the establishment of tumors (Fig. 7a) and increase animal survival (Fig. 7b) to protect mice from secondary antigen administration to tumor cells. This result demonstrates that the peptides of the present invention are still functional for secondary antigen administration to tumor cells that can induce a sustained anti-tumor response and demonstrate the development of an antigen-specific adaptive immune response.

Example 9. Anti-tumor effect of L-2-like peptides in a metastasis model of Lewis carcinoma.

8-10 week old C57Bl / 6 female mice were used for this assay (n = 8 animals per experimental group). 250,000 mouse lung cancer 3LLD122 cells were inoculated into the hind footpad of these animals. After 7 days, a dose of 4 mg / kg body weight of L-2 similar peptide was injected subcutaneously. When the diameter of the tumor reached 8 mm, the initial tumor was surgically removed from the carrier's leg. Mice were sacrificed on day 21 after this surgical procedure. The weight of the lungs was measured to show the amount of metastasis in the lungs. The results shown in FIG. 8 indicate that the L-2-like peptides of the present invention can reduce metastatic tumorigenesis.

Example 10. Effect of L-2-like peptides on growth of tumor cells.

In this assay, TC-1, H-125 (arsenic human lung cancer cells) and L929 mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with fetal bovine serum (Gibco) And inoculated into 96-well culture plates (Costar) at 2 x 10 &lt; ⁴ &gt; cells / ml. After 24 hours, the peptide was added to the culture in the range of 9 [mu] M to 300 [mu] M. Plates were inoculated for 72 h under 5% CO ₂ , then the assay was expressed as crystal violet. The plate was extensively washed with tap water and the plate was read at 562 nm. The results are shown in Fig. Apoptotic peptides with significant anti-proliferative effects in vitro were used as positive controls (Perea, S., et al. (2004) Antitumor Effect of a Novel Proapoptotic Peptide that Impairs the Phosphorylation by the Protein Kinase 2 Cancer Research 64: 7127-7129). The results obtained demonstrate that L-2 peptide produces a dose-dependent anti-proliferative effect in TC-1 and H-125 cells. However, the effect of the peptide of the present invention was not detected in the mouse fibroblast L929 cell line. These results demonstrate that the peptides of the present invention exert selective cytotoxic effects on tumor cells ex vivo.

SEQUENCE LISITING <110> Centro de Ingenieria Genetica y Biotecnologia <120> "IMMUNOMODULATERY AND ANTI-TUMOR PEPTIDES". <130> Anti-tumoral <140> <141> <150> CU 2006-0047 <151> 24-02-2006 <160> 13 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 33 by Alanine <400> 1 His Ala Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 39 by Alanine <400> 2 His Tyr Arg Ile Lys Pro Thr Ala Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 43 by Alanine <400> 3 His Tyr Arg Ile Lys Pro Thr Phe Arg Arg Leu Ala Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 51 by Alanine 51 <400> 4 His Tyr Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Ala              20 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 34 by Alanine <400> 5 His Tyr Ala Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 40 by Alanine <400> 6 His Tyr Arg Ile Lys Pro Thr Phe Ala Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 50 by Alanine <400> 7 His Tyr Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Ala Trp              20 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a 32 to 51 of the LALF protein, modified by       substituting the a.a. 39 and 43 by Alanine <400> 8 His Tyr Arg Ile Lys Pro Thr Ala Arg Arg Leu Ala Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a 32 to 51 of the LALF protein, modified by       substituting the a.a. 33 and 39 by Alanine <400> 9 His Ala Arg Ile Lys Pro Thr Ala Arg Arg Leu Lys Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 10 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 33 and 43 by Alanine <400> 10 His Ala Arg Ile Lys Pro Thr Phe Arg Arg Leu Ala Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 11 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 33, 39 and 43 by Alanine <400> 11 His Ala Arg Ile Lys Pro Thr Ala Arg Arg Leu Ala Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Trp              20 <210> 12 <211> 20 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1) <223> Description of the Artificial Sequence: Peptide       from a.a. 32 to 51 of the LALF protein, modified by       substituting the a.a. 33, 39, 43 and 51 by Alanine <400> 12 His Ala Arg Ile Lys Pro Thr Ala Arg Arg Leu Ala Trp Lys Tyr Lys   1 5 10 15 Gly Lys Phe Ala              20 <210> 13 <211> 21 <212> PRT <213> Limulus polyphemus <220> <221> PEPTIDE <222> (1) <223> Peptide from a.a. 32 to 52 of the LALF protein <400> 13 His Tyr Arg Ile Lys Pro Thr Phe Arg Arg Leu Lys Trp Lys Lys Tyr   1 5 10 15 Lys Gly Lys Phe Trp              20

Claims (8)

Amino acid sequence SEQ. ID. NO. An anti-tumor consisting of an amino acid sequence selected from 1 to 4, lacking LPS-binding and heparin binding capacity, and derived from the 32-51 region of the LALF protein and having immunomodulating ability. delete One or more of the peptides according to claim 1; And a pharmaceutical composition for the treatment or prevention of cancer comprising a cosmetic or pharmaceutical acceptable excipient. 4. The pharmaceutical composition of claim 3, wherein the composition further comprises an immunogen. 5. The method of claim 4, wherein the immunogenic agent is a proteinaceous carrier or viral particle of bacterial origin; Peptidic immunogens, ganglioside immunogens or proteinaceous immunogens. delete 4. A pharmaceutical composition according to claim 3 comprising an effective amount of said peptide for stimulating an innate immune response in the human body. 4. The pharmaceutical composition according to claim 3, which is applied to inhibit metastasis.

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